Compositions for improving cell viability and methods of use thereof

ABSTRACT

This invention relates to methods and compositions for use improving cell viability, particularly neural cell viability, and more particularly to methods and compositions for use improving cell viability by reducing reactive oxygen metabolite-mediated oxidative damage in a cell, regulating redox homeostasis in a cell, or reducing mitochondrial dysfunction in a cell. The invention further relates to the administration of the bile acid tauroursodeoxycholic acid (TUDCA) in combination with phenylbutyric Acid (PBA) to improve cell viability, and treat at least one symptom associated with, prevent the time of onset of, or slow the development of a disease related to oxidative stress.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.14/140,083, filed on Dec. 24, 2013, which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/804,690, filed on Mar. 24,2013, the entire contents of which are hereby incorporated by reference.

TECHNICAL FIELD

This invention relates to methods and compositions for use in improvingcell viability, particularly neuronal viability, and more particularlyto methods and compositions for use in improving cell viability throughthe reduction of reactive oxygen metabolite-mediated oxidative damage ina cell, regulating redox homeostasis in a cell, or reducingmitochondrial dysfunction in a cell. This invention relates to the fieldof pharmaceutical treatments, and more particularly to the treatment ofAlzheimer's disease and other Amyloidosis related pathology.

BACKGROUND

Neurodegenerative diseases of the central nervous system (CNS) causeprogressive loss of neuronal structure and function and are devastatingdiseases for affected patients and their families. Among theseneurodegenerative diseases are, for example, Multiple Sclerosis (MS),Parkinson's disease, Alzheimer's disease, Huntington's disease,amyotrophic lateral sclerosis (ALS) and stroke. Due to the complexity ofthe CNS, many of these diseases are only poorly understood to date.

Alzheimer's disease is the most prevalent neurodegenerative disease andone of the largest medical problems in the United States. In 2012, anestimated 5.4 million Americans were suffering from the disease and itwas the sixth leading cause of death. As increasing age is the largestrisk factor for Alzheimer's, the number of afflicted is expected to riseto 7.1 million by 2025 as the population of the United States ages.Other risk factors include certain genetic mutations, diabetes, andinflammation.

Alzheimer's disease is characterized by the aggregation of amyloid betainto plaques and the formation of neurofibrillary tangles mediated byvarious forms of phosphorylated tau protein. Some major symptoms of thedisease include memory loss, challenges in completing and planningroutine tasks, confusion with time or place, problems with words orspeaking, and personality changes.

Alzheimer's is the most common member of a broad class of dementias,many of which are thought to be mediated by amyloid plaques, amyloidoligomer formation, and/or phosphorylated tau protein. These diseasesinclude, but are not limited to, Pick's Disease, Multi-Infarct Dementia,Creutzfeldt-Jakob's Disease, Dementia with Lewy bodies, Mixed dementia,and Frontotemporal dementia.

Currently approved drugs used in the treatment of Alzheimer's diseaseeither block NMDA-type glutamate receptors or are acetyl cholinesteraseinhibitors; the latter only modestly effective for about 6-12 months inonly fifty percent of patients and only under certain cognitive tests.Both classes of drugs are based on a model of increasing neuralexcitement globally to the generally depressed brain, making them proneto cause many side effects and doing nothing to alter disease pathology.While several drug classes are known or have been suggested for treatingneurodegenerative diseases, effective therapies are scarce ornon-existent. Thus, there is need for improved therapies for treatingneurodegenerative diseases.

SUMMARY

At least in part, the present invention is based on the discovery thatcompositions comprising a bile acid (e.g., tauroursodeoxycholic acid(TUDCA)) in combination with a phenylbutyric acid (PBA) (e.g.,4-phenylbutyric acid (4-PBA)) significantly reduce reactive oxygenmetabolite-mediated oxidative damage in a cell, resulting in improvedcell viability (e.g., neuronal viability). As discussed in the followingexamples, the compounds TUDCA and 4-PBA were evaluated individually andin combination as a protectant against hydrogen peroxide-induced cellapoptosis. Hydrogen peroxide-induced cell apoptosis is thought to becaused through the altering of redox homeostasis, the overproduction ofreactive oxygen species, and mitochondrial dysfunction. The examplesdemonstrate that TUDCA and 4-PBA, when measuring cell viability and/orcell death, have a greater than additive effect in protecting the cellsagainst hydrogen peroxide exposure. This surprising discovery suggeststhat these drugs synergistically augment each other's efficacy inreducing the aforementioned pathologies.

This invention provides methods and compositions for use in improvingcell viability, particularly neuronal viability, and more particularlyto methods and compositions for use improving cell viability by reducingreactive oxygen metabolite-mediated oxidative damage in a cell,regulating redox homeostasis in a cell, or reducing mitochondrialdysfunction in a cell. The invention further relates to theadministration of a bile acid (e.g., tauroursodeoxycholic acid (TUDCA))in combination with a phenylbutyric acid (e.g., 4-PBA) to improve cellviability and to treat at least one symptom associated with, prevent thetime of onset of, or slow the development of a disease related tooxidative stress.

The invention further provides a novel approach for reducing neuronaloxidative stress and for treating at least one symptom associated with,prevent the time of onset of, or slow the development of a diseaserelated to oxidative stress, including but not limited toneurodegenerative diseases (e.g. Alzheimer's Disease (AD), Huntington'sdisease (HD), Parkinson's disease (PD), Amyotrohic Lateral Sclerosis,Pick's Disease, Multi-Infarct Dementia, Creutzfeldt-Jakob's Disease,Dementia with Lewy bodies, Frontotemporal dementia) comprisingadministration of a composition comprising a combination of a bile acid(e.g., tauroursodeoxycholic acid (TUDCA)) and a phenylbutyric acid(e.g., 4-PBA) or analogs, derivatives, pharmacological equivalents, orsalts thereof in a pharmaceutical composition or formulation.

In one aspect, the disclosure provides a method for reducing reactiveoxygen metabolite-mediated oxidative damage in a cell, the methodcomprising contacting the cell with a bile acid, or a pharmaceuticallyacceptable salt, analog, derivative, or prodrug thereof; and aphenylbutyric acid (PBA), or a pharmaceutically acceptable salt, analog,derivative, or prodrug thereof. In one embodiment, the reactive oxygenmetabolite-mediated oxidative damage is hydrogen peroxide (H₂O₂)mediated damage.

In another aspect, the disclosure provides a method of regulating redoxhomeostasis in a cell, the method comprising contacting the cell with abile acid, or a pharmaceutically acceptable salt, analog, derivative, orprodrug thereof; and a phenylbutyric acid (PBA), or a pharmaceuticallyacceptable salt, analog, derivative, or prodrug thereof.

In yet another aspect, the disclosure provides a method of reducingmitochondrial dysfunction in a cell, the method comprising contactingthe cell with a bile acid, or a pharmaceutically acceptable salt,analog, derivative, or prodrug thereof; and a phenylbutyric acid (PBA),or a pharmaceutically acceptable salt, analog, derivative, or prodrugthereof.

In one or more embodiments, the bile acid is selected from the groupconsisting of tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid(UDCA), chenodeoxycholic acid, cholic acid, hyodeoxycholic acid,deoxycholic acid, 7-oxolithocholic acid, lithocholic acid,iododeoxycholic acid, iocholic acid, taurochenodeoxycholic acid,taurodeoxycholic acid, glycoursodeoxycholic acid, taurocholic acid,glycocholic acid, or an analog, derivative, or derivative thereof. Insome embodiments, the bile acid is selected from the group consisting oftauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA).

In some embodiments, the cell is contacted with a bile acid at aconcentration of about 80 μM to about 120 μM.

In one or more embodiments, the PBA is 4-phenylbutyric acid,glycerly(Tri-4-PBA), phenyl acetic acid, 2-POAA-OMe, 2-POAA-NO2, 2-NOAAor a pharmaceutically acceptable salt, analog, derivative, or prodrugthereof.

In some embodiments, the cell is contacted a with phenylbutyric acid ata concentration of about 0.8 mM to about 1.2 mM. In some aspects, thecell is a mammalian cell. In an embodiment, the cell is a human cell. Inanother embodiment, the cell is a neuron.

In certain aspects, the disclosure provides a method of treating aneurodegenerative disease associated with reactive oxygenmetabolite-mediated oxidative damage in a subject, the method comprisingidentifying a subject experiencing a neurodegenerative diseaseassociated with reactive oxygen metabolite-mediated oxidative damage;administering to said subject a bile acid, or a pharmaceuticallyacceptable salt, analog, derivative, or prodrug thereof; and aphenylbutyric acid (PBA), or a pharmaceutically acceptable salt, analog,derivative, or prodrug thereof, wherein the amount of PBA administeredin combination with a bile acid is reduced by 10% to 55% compared toadministration of PBA alone.

In yet another aspect, the disclosure provides a method of treating aneurodegenerative disease in a subject in need thereof, the methodcomprising identifying a subject with at least one copy of the APOEε4allele, administering to said subject a composition comprising a bileacid, or a pharmaceutically acceptable salt, analog, derivative, orprodrug thereof; and a phenylbutyric acid (PBA), or a pharmaceuticallyacceptable salt, analog, derivative, or prodrug thereof to thereby treatthe neurodegenerative disease.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph demonstrating the ability of TUDCA and PBA incombination to increase cell viability evaluated by PrestoBlue afterHydrogen Peroxide exposure in rat cortical neuron culture. **** denotessignificance of p<0.0001 against control. ## denotes significance ofp<0.01 against peroxide exposure. # denotes significance of p<0.05against peroxide exposure. The combination provides significantprotection while each drug individually does not provide significantprotection. NMDA exposure at 300 uM is used as a negative control. DFX(not shown) was not included in the plot as it is thought to interferewith PrestoBlue measurement and provided erratic results.

FIG. 2 is a graph demonstrating the ability of TUDCA and PBA incombination to ameliorate cell death evaluated by LDH after HydrogenPeroxide (H₂O₂) exposure in rat cortical neuron culture. **** denotessignificance of p<10-4 against control. ## denotes significance ofp<0.01 against peroxide exposure. # denotes significance of p<0.05against peroxide exposure. The combination provides significantprotection while each drug individually does not provide significantprotection. NMDA exposure at 300 uM is used as a negative control whileDFX is a positive control in the LDH study.

FIG. 3 demonstrates the chemical structure of TUDCA (formula I) withlabeled carbons to assist in understanding where substitutions can bemade.

FIG. 4 demonstrates the chemical structure of UDCA (formula II) withlabeled carbons to assist in understanding where substitutions can bemade.

FIG. 5 demonstrates the chemical structure of PBA stabilized by sodiumion (formula III). Derivatives available for use under this inventionare described in the background.

DETAILED DESCRIPTION

Diabetes, inflammation, increasing age, specific genetic variants, andmany other conditions are risk factors for Alzheimer's disease. A commonthread between these conditions is that they are all associated withincreased free radical production, an imbalance in redox homeostasis,and/or increased free-radical mediated damage to cells and tissue.Superoxide and other free radicals have been implicated in increasingthe amount of pathogenic amyloid proteins and increasing aggregationinto amyloid plaques, oligomers, and other species. Without being boundby theory, this aggregation has been attributed by some to a processinvolving oxidation of an amino acid of the amyloid chain, especially atthe Methionine 35 residue.

Incorporation of amyloid protein groups into the mitochondria has beenimplicated in the release of cytochrome c, oxygen radicals, and otherfree radicals. These radicals have been shown to increase theendoplasmic reticulum stress response and also increase the aggregationof amyloid protein.

Recent reports suggest that reactive oxygen metabolites may be involvedin the pathology of Alzheimer's disease. Oxygen radicals, as well aslipid and nitrogenous radicals, are precursors in BAX, Cytochrome C, andJNK mediated pathways of apoptosis. Evidence also suggests that thealteration in redox state resultant from these species causes anincrease in proteolytic activity critical in the pathology of manydiseases caused at least in part by abnormal protein processing.Alteration of the cell redox state also causes a reduction in majorantioxidant species such as glutathione (GSH) and vitamin E.

Reactive Oxygen Metabolite-Mediated Damage

Recent studies have also begun to implicate reactive oxygenmetabolite-mediated damage as one of the major factors inneurodegenerative disease. Hydrogen peroxide, for example, has beenshown to affect the mitochondria, the proteolytic state of the cell, theproduction of amyloid beta, redox homeostasis, and many apoptoticpathways within the cell. Hydrogen peroxide has also been shown to reactwith metal ions such as iron and copper to form more reactive oxygenspecies through Fenton chemistry. The decrease in antioxidants such asvitamin E and Glutathione in multiple neurodegenerative pathologiessuggests an increase in oxidative damage in these diseases.

PINK1 and Parkin (Parkinson protein 2, E3 ubiquitin protein ligase) arethought to regulate quality control in mitochondria and mutations ofthese genes can be causative for Parkinson's Disease. Studies have shownthat transcription of Parkin increases following the introduction ofhydrogen peroxide to a cell. MPTP((1-methyl-4-phenyl-1,2,3,6-tetra/hydropyridine) which inducesParkinson's like symptoms also results directly in the production ofhydrogen peroxide. Some studies have demonstrated up to a 90% increasein hydrogen peroxide levels in this disease.

In Alzheimer's disease, amyloid beta has been shown to directly producehydrogen peroxide when contacted with metal ions such as iron andcopper, which are found in higher concentrations in the brains ofpatients suffering from the disease. Hydrogen peroxide may also increasethe processing of amyloid beta into a pathogenic form. Hydrogen peroxideand reactive oxygen-metabolite mediated pathways are also thought to bea major route of cell death in this disease.

Huntington's disease also shows proteolytic products likely to be causedby hydrogen peroxide, as well as widespread mitochondrial dysfunction.The increase in 3-hydroxykyneurine has been implicated as a majorpathological step in Huntington's disease and is known to directlyinduce hydrogen peroxide and hydroxyl radical production.

Hydrogen peroxide has also been shown to be a potential mediator in thepathology of ALS. Scavengers of hydrogen peroxide have been consideredas therapeutics in this field.

Regulation of Redox Homeostasis

Redox homeostasis refers to the attempt of a cell to manage reductiveand oxidative species in the cell to maintain a constant redox state.Disruptions in redox homeostasis may change free energy requirements andallow processes to take place in cells that would not occur undernon-pathologic conditions. Some processes that may be caused by failureof regulation of redox homeostasis include the aggregation of amyloidbeta into plaques characteristic of Alzheimer's disease, the Lewy Bodiesof Parkinson's disease and Dementia with Lewy Bodies, the senile plaquesof Huntington's disease, the plaques including amyloid plaques that havebeen found in ALS, and the disorders of tauopathies. Under typicalphysiological conditions these proteins will not aggregate, butincreased concentrations as well as atypical redox states are thought toalter the free energy of aggregation resulting in the observed plaquesin these pathologies. Altered redox state can also produce free radicalswhich interact with mitochondrial pathways to induce apoptosis. A needtherefore exists to develop an agent that can aid in regulation of theredox state of a cell. Since Hydrogen Peroxide induces altered redoxstate of a cell it can be used to cause redox-mediated damage to a cell.

Mitochondrial Dysfunction

Mitochondrial dysfunction is widespread in neurodegenerative disease. InAlzheimer's disease, the mitochondrial membrane potential of cells ismarkedly reduced, glucose metabolism by the mitochondria is impaired,and the permeability of the mitochondria is increased. Mitochondria havebeen observed to mediate multiple apoptotic pathways resulting inneuronal death in Alzheimer's disease.

PINK1 and Parkin are both mitochondrial quality control proteins.Mutations or lack of these proteins is strongly linked to Parkinson'sdisease. MPTP, a molecule used to induce permanent symptoms ofParkinson's, acts through the disruption of complex I of themitochondria, causing mitochondrial dysfunction, alteration of the redoxstate of the cell, and apoptosis.

It has been directly shown in cell culture that the mutant Huntingtingene and its resultant protein, thought to be the primary mediator ofHuntington's disease, results in a loss of membrane potential anddecreased expression of critical oxidative phosphorylation genes in themitochondria. Huntington's disease pathology has also been linked to adecrease in the number of mitochondria present in the central nervoussystem.

Mitochondrial dyslocalization, energy metabolism impairment, andapoptotic pathways are thought to mediate Amyotrophic lateral sclerosis.Mitochondria from affected tissues have also been shown to overproducereactive oxygen metabolites and leak them to the cytosol.

In many neurodegenerative diseases, mitochondria overproduce freeradicals, cause a reduction in energy metabolism, have increasedpermeability, have decreased membrane potential, have decreasedantioxidants, leak metal ions into the cell, alter the redox state ofthe cell, and lead the cell down pro-apoptotic pathways. A needtherefore exists for agents that can alter and reduce mitochondrialdysfunction mechanisms. APOEε4 Allele

The genetics of Alzheimer's disease is complex. Mutations in at leastfour genetic loci are associated with inherited susceptibility to AD(i.e., familial AD). Three genes have been associated with early onsetAD: APP [β-amyloid precursor protein on chromosome 21], PS1(presenilin 1) and PS2 (presenilin 2). The ε4 allele of theapolipoprotein E (APOE) gene on chromosome 19 has been associated withlate onset AD. The association of APOEε4 with AD appears to be strongestin individuals with an onset prior to 70 years of age and weakens withadvanced age. Patients who present with at least one copy of the APOE ε4allele have been shown to have particularly elevated levels of peroxidemetabolites as well as mitochondrial damage. Treatment to this patientpopulation may be particularly effective.

In certain aspects, the disclosure contemplates a method of treating aneurodegenerative disease in a subject in need thereof, the methodcomprising identifying a subject with at least copy of the APOEε4allele, administering to said subject a composition comprising a bileacid, or a pharmaceutically acceptable salt, analog, derivative, orprodrug thereof; and a phenylbutyric acid (PBA), or a pharmaceuticallyacceptable salt, analog, derivative, or prodrug thereof to thereby treatthe neurodegenerative disease.

In certain aspect, the invention provides methods comprising contactinga cell with a bile acid. As used herein, “bile acid” (e.g., aqueoussoluble bile acid derivatives, bile acid salts, or bile acid conjugatedwith an amine) refers to naturally occurring surfactants having anucleus derived from cholanic acid substituted with a 3α-hydroxyl groupand optionally with other hydroxyl groups as well, typically at the C₆,C₇ or C₁₂ position of the sterol nucleus. Bile acid derivatives include,but are not limited to derivatives formed at the hydroxyl and carboxylicacid groups of the bile acid with other functional groups including butnot limited to halogens and amino groups. Soluble bile acid may includean aqueous preparation of a free acid form of bile acid combined withone of HCl, phosphoric acid, citric acid, acetic acid, ammonia, orarginine.

In certain aspects, the methods of the present invention comprisecontacting a cell with a bile acid, or a pharmaceutically acceptablesalt, analog, derivative, or prodrug thereof. In one or moreembodiments, the bile acid is tauroursodeoxycholic acid (TUDCA) as shownin formula I (FIG. 3), with labeled carbons to assist in understandingwhere substitutions may be made.

In one or more embodiments, the bile acid ursodeoxycholic acid (UDCA) asshown in formula II (FIG. 4), with labeled carbons to assist inunderstanding where substitutions may be made.

Physiologically related bile acid derivatives include any combination ofsubstitutions of hydrogen at position 3 or 7, a shift in thestereochemistry of the hydroxyl group at positions 3 or 7, andpharmaceutically acceptable salts, solvates or amino acid conjugatesthereof of TUDCA or UDCA.

In some embodiments, the bile acid is a TUDCA compound of formula IV:

wherein R is —H or C₁-C₄ alkyl;R₁ is —CH₂—SO₃R₃ and R₂ is —H; or R₁ is —COOH and R₂ is —CH₂—CH₂—CONH₂,—CH₂—CONH₂, —CH₂—CH₂—SCH₃ or —CH₂—S—CH₂—COOH; andR₃ is —H or the residue of a basic amino acid, or a pharmaceuticallyacceptable analog, derivative, prodrug thereof, or a mixture thereof,

This invention also contemplates the use of bile acids in addition toTUDCA and UDCA, including, for example, chenodeoxycholic acid (alsoreferred to as “chenodiol” or “chenic acid”), cholic acid,hyodeoxycholic acid, deoxycholic acid, 7-oxolithocholic acid,lithocholic acid, iododeoxycholic acid, iocholic acid,taurochenodeoxycholic acid, taurodeoxycholic acid, glycoursodeoxycholicacid, taurocholic acid, glycocholic acid, cholic acid, or an analog,derivative, or derivative thereof.

In certain aspect, the invention provides methods comprising thecontacting a cell with a phenylbutyric acid. Phenylbutyric acid (PBA)(FIG. 4, sodium salt) is a HDAC2 (Histone Deacetylase 2) inhibitor.Uptake of PBA and derivatives results in differential gene expressionwhich has been shown to have a variety of effects. PBA has also beenshown to act as a chemical chaperone with a variety of effects. Oneeffect is the decrease in production of pathogenic amyloid protein.Another effect is increased neuroplasticity. PBA has also been shown toimprove biliary excretion. PBA is however known to be cytotoxic tomultiple different cell types.

Physiologically related phenylbutyric acid (PBA) species include anysubstitutions for Hydrogens with Deuterium. Any salts, solvates,conjugates and pharmacologically related compounds will also beconsidering physiologically related. Also all phenylbutyric acidderivatives described in Prior Art will be considered physiologicallyrelated. Other HDAC2 inhibitors will also be considered as substitutesfor phenylbutyrate. Phenylacetic acid, the active metabolite of PBA mayalso be considered as a substitute.

In one or more embodiments the PBA compound is 4-Phenylbutyric acid(4-PBA). 4-PBA is a low molecular weight aromatic carboxylic acid. 4-PBAis defined herein as encompassing not only 4-Phenybutyric acid as a freeacid but also its derivatives and physiologically acceptable saltsthereof. Especially, “4-Phenylbutyric acid” or “4-PBA” is also definedas its free acid, but also as being in the form of a pharmaceuticallyacceptable salt, co-crystal, polymorph, hydrate, solvate or pro-drug of4-phenylbutyric acid. Most preferably, “4-Phenylbutyric acid” or “4-PBA”is either the free acid or a pharmaceutically acceptable salt of 4-PBA,such as its sodium salt. Analogs of 4-PBA included, for example,Glycerly(Tri-4-PBA), Phenyl Acetic Acid, 2-POAA-OMe, 2-POAA-NO2, 2-NOAA.Physiologically acceptable salts of 4-phenylbutyrate, include, forexample sodium, potassium, magnesium or calcium salts.

Pharmaceutical Compositions and Methods of Administration

In certain aspects, the methods described herein include the manufactureand use of pharmaceutical compositions and medicaments that includecompounds identified by a method described herein as active ingredients.Also included are the pharmaceutical compositions themselves.

In some instances, the compositions disclosed herein can include othercompounds, drugs, and/or agents used for the treatment ofneurodegenerative disease. For example, in some instances, therapeuticcompositions disclosed herein can be combined with one or more (e.g.,one, two, three, four, five, or less than ten) compounds.

In some instances, the compositions disclosed herein can include othercompounds including COX2 inhibitors, asthma drugs, diabetes drugs, otherantioxidants, acetyl cholinesterase inhibitors (e.g., donepezil,tacrine, rivastigmine, galantamine, physostigmine, neostigmine,Huperzine A, icopezil (CP-118954,5,7-dihydro-3-[2-[1-(phenylmethyl)-4-piperidinyl]ethyl]-6H-pyrrolo-[4,5-f]-1,2-benzisoxazol-6-onemaleate), ER-127528(4-[(5,6-dimethoxy-2-fluoro-1-indanon)-2-yl]methyl-1-(3-fluorobenzyl)piperidine hydrochloride), zanapezil (TAK-147;3-[1-(phenylmethyl)piperidin-4-yl]-1-(2,3,4,5-tetrahydro-1H-1-benzazepin-8-yl)-1-propanefumarate), Metrifonate (T-588;(−)-R-a-[[2-(dimethylamino)ethoxy]methyl]benzo[b]thiophene-5-methanolhydrochloride), FK-960(N-(4-acetyl-1-piperazinyl)-p-fluorobenzamide-hydrate), TCH-346(N-methyl-N-2-pyropinyldibenz[b,f] oxepine-10-methanamine), SDZ-220-581((S)-a-amino-5-(phosphonomethyl)-[1,1′-biphenyl]-3-propionic acid), andcombinations thereof), NMDA receptor antagonists (e.g., memantine,neramexane, rimantadine, or amantadine, lipoxygenase inhibitors,leukotriene inhibitors, coconut oil, other HDAC inhibitors, statins,amphetamines, other MAO inhibitors, metal chelators, BACE1-inhibitors,antibodies to amyloid beta, gamma-secretase modulators, amyloid clearingagents, phosphorylated tau antibodies, Aβ inhibitors, Aβ plaque removalagents, inhibitors of Aβ plaque formation, inhibitors of amyloidprecursor protein processing enzymes, β-amyloid converting enzymeinhibitors, β-secretase inhibitors, γ-secretase modulators, nerve growthfactor agonists and neurofibrillary tangle clearing agents).

In some instances, compositions disclosed herein can be formulated foruse as or in pharmaceutical compositions. Such compositions can beformulated or adapted for administration to a subject via any route,e.g., any route approved by the Food and Drug Administration (FDA).Exemplary methods are described in the FDA's CDER Data Standards Manual,version number 004 (which is available atfda.give/cder/dsm/DRG/drg00301.htm). The pharmaceutical compositions maybe formulated for oral, parenteral, or transdermal delivery. Thecompound of the invention may also be combined with other pharmaceuticalagents. In some aspects, the invention provides kits that include theTUDCA and PBA compounds used in the invention. The kit may also includeinstructions for the physician and/or patient, syringes, needles, box,bottles, vials, etc. In an aspect, the invention provides methods andagents that are useful in preventing or treating neurodegenerativedisease, including Multiple Sclerosis (MS), Parkinson's disease,Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis(ALS), stroke, Pick's Disease, Multi-Infarct Dementia,Creutzfeldt-Jakob's Disease, Dementia with Lewy bodies, Mixed dementia,Frontotemporal dementia, and associated diseases. In particular, theinvention provides agents or pharmaceutical compositions that can beused to treat or prevent Alzheimer's disease and other amyloidosisrelated pathologies, and prevent complications of these conditions.

In some instances, pharmaceutical compositions can include an effectiveamount of a bile acid and a phenylbutyric acid as described above. Theterms “effective amount” and “effective to treat,” as used herein, referto an amount or a concentration of one or more drugs for a period oftime (including acute or chronic administration and periodic orcontinuous administration) that is effective within the context of itsadministration for causing an intended effect or physiological outcome.

In some instances, pharmaceutical compositions can include of a bileacid (e.g., TUDCA), a phenylbutyric acid (e.g., 4-PBA), and anypharmaceutically acceptable carrier, adjuvant and/or vehicle. In someinstances, pharmaceuticals can further include one or more additionaltherapeutic agents in amounts effective for achieving a modulation ofdisease or disease symptoms.

Pharmaceutical compositions typically include a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier oradjuvant” refers to a carrier or adjuvant that may be administered to apatient, together with a compound of this invention, and which does notdestroy the pharmacological activity thereof and is nontoxic whenadministered in doses sufficient to deliver a therapeutic amount of thecompound. As used herein the language “pharmaceutically acceptablecarrier” includes saline, solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.

The pharmaceutical compositions of this invention may contain anyconventional non-toxic pharmaceutically-acceptable carriers, adjuvantsor vehicles. In some cases, the pH of the formulation may be adjustedwith pharmaceutically acceptable acids, bases or buffers to enhance thestability of the formulated compound or its delivery form. The termparenteral as used herein includes subcutaneous, intracutaneous,intravenous, intramuscular, intra-articular, intraarterial,intrasynovial, intrasternal, intrathecal, intralesional and intracranialinjection or infusion techniques.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration.

Pharmaceutical compositions can be in the form of a solution or powderfor inhalation and/or nasal administration. Such compositions may beformulated according to techniques known in the art using suitabledispersing or wetting agents (such as, for example, Tween 80) andsuspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are mannitol, water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose, any blandfixed oil may be employed including synthetic mono- or diglycerides.Fatty acids, such as oleic acid and its glyceride derivatives are usefulin the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,or carboxymethyl cellulose or similar dispersing agents which arecommonly used in the formulation of pharmaceutically acceptable dosageforms such as emulsions and or suspensions. Other commonly usedsurfactants such as Tweens or Spans and/or other similar emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

Pharmaceutical compositions can be orally administered in any orallyacceptable dosage form including, but not limited to, capsules, tablets,emulsions and aqueous suspensions, dispersions and solutions. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions and/or emulsions are administered orally, the activeingredient may be suspended or dissolved in an oily phase is combinedwith emulsifying and/or suspending agents. If desired, certainsweetening and/or flavoring and/or coloring agents may be added.

Alternatively or in addition, pharmaceutical compositions can beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other solubilizing or dispersingagents known in the art.

In some embodiments, the present disclosure provides methods for using acomposition comprising a bile acid (e.g., TUDCA) and a phenylbutyricacid (e.g., 4-PBA), including pharmaceutical compositions, (indicatedbelow as ‘X’) disclosed herein in the following methods:

Substance X for use as a medicament in the treatment of one or morediseases or conditions disclosed herein (e.g., neurodegenerativedisease, referred to in the following examples as ‘Y’). Use of substanceX for the manufacture of a medicament for the treatment of Y; andsubstance X for use in the treatment of Y.

In some instances, therapeutic compositions disclosed herein can beformulated for sale in the US, import into the US, and/or export fromthe US.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Dosage

In some aspects of the invention, TUDCA (Tauroursodeoxycholic Acid) andPBA (4-Phenylbutyrate), are individually administered (i.e., separatedosage forms). In some embodiments, TUDCA is administered in amount ofabout 10 mg/kg body weight, about 15 mg/kg body weight, about 20 mg/kgbody weight, about 30 mg/kg body weight, about 40 mg/kg body weight, orabout 40 mg/kg body weight. In some embodiments, PBA is administered inamount of about 10 mg/kg body weight, about 30 mg/kg body weight, about50 mg/kg body weight, about 100 mg/kg body weight, about 200 mg/kg bodyweight, or about 400 mg/kg body weight. The compounds can beadministered separately or together, including as a part of a regimen oftreatment.

The invention further provides dosing regimens, such that the TUDCAand/or PBA dosage forms are administered, separately or together, as asingle daily dosage, on a daily basis, a weekly basis or some otherbasis. Further, the patient may receive the specific dosage over aperiod of weeks, months, or years. For example, 1 week, 2 weeks, 3weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3years, 4 years, 5 years and the like.

Advantages of the compositions of the invention, include, (1) smallersolid PBA dosage form size; and (2) smaller doses of PBA required toobtain the same pharmacological effect. Thus, in certain aspects, thisinvention provides improved methods for treating neurodegenerativedisease that comprise administering a PBA, in combination with a bileacid, in a reduced dosage amount compared with treatment ofneurodegenerative diseases with PBA (e.g., compared with standard dosingamounts of PBA). In some embodiments, the amount of PBA administered incombination with a bile acid is reduced by about 10%, about 15%, about20%, about 25%, about 30%, about 40%, about 45%, about 50%, or about 55%compared to the dosage amount used what PBA is administered alone.

Methods of Treatment

The methods described herein include methods for the treatment ofdisorders associated with cellular oxidative stress (e.g., reactiveoxygen metabolite-mediated oxidative damage in a cell, redox imbalancein a cell, or mitochondrial dysfunction in a cell). In some embodiments,the disorder is a neurodegenerative disease (e.g., Alzheimer's disease(AD), Huntington's disease (HD), Parkinson's disease (PD), AmyotrohicLateral Sclerosis). Generally, the methods include administering atherapeutically effective amount of a bile acid (e.g., TUDCA) incombination with a phenylbutyric acid (e.g., 4-PBA) as described herein,to a subject (e.g., a mammalian subject, e.g., a human subject) who isin need of, or who has been determined to be in need of, such treatment.

In some instances, methods can include selection of a human subject whohas or had a condition or disease. In some instances, suitable subjectsinclude, for example, subjects who have or had a condition or diseasebut that resolved the disease or an aspect thereof, present reducedsymptoms of disease (e.g., relative to other subjects (e.g., themajority of subjects) with the same condition or disease), and/or thatsurvive for extended periods of time with the condition or disease(e.g., relative to other subjects (e.g., the majority of subjects) withthe same condition or disease), e.g., in an asymptomatic state (e.g.,relative to other subjects (e.g., the majority of subjects) with thesame condition or disease).

The methods disclosed herein can be applied to a wide range of species,e.g., humans, non-human primates (e.g., monkeys), horses, cattle, pigs,sheep, deer, elk, goats, dogs, cats, rabbits, guinea pigs, hamsters,rats, and mice.

The terms “treat”, “treating”, “treatment”, etc., as applied to anisolated cell, include subjecting the cell to any kind of process orcondition or performing any kind of manipulation or procedure on thecell. As applied to a subject, the term “treating” refer to providingmedical or surgical attention, care, or management to an individual. Theindividual is usually ill or injured, or at increased risk of becomingill relative to an average member of the population and in need of suchattention, care, or management.

In some embodiments, the term “treating” and “treatment” refers toadministering to a subject an effective amount of a composition, e.g., acomposition comprising a bile acid and a phenylbutyric acid, so that thesubject has a reduction in at least one symptom of the disease or animprovement in the disease, for example, beneficial or desired clinicalresults. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation of one or moresymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. Treating canrefer to prolonging survival as compared to expected survival if notreceiving treatment. Thus, one of skill in the art realizes that atreatment may improve the disease condition, but may not be a completecure for the disease. In some embodiments, treatment can be “prophylaxictreatment, where the subject is administered a composition as disclosedherein (e.g., a composition comprising a bile acid and a phenylbutyricacid) to a subject at risk of developing a neurodegenerative disease asdisclosed herein. In some embodiments, treatment is “effective” if theprogression of a disease is reduced or halted.

The term “subject,” as used herein, refers to any animal. In someinstances, the subject is a mammal. In some instances, the term“subject”, as used herein, refers to a human (e.g., a man, a woman, or achild).

In some instances, subject selection can include obtaining a sample froma subject (e.g., a candidate subject) and testing the sample for anindication that the subject is suitable for selection. In someinstances, the subject can be confirmed or identified, e.g. by a healthcare professional, as having had or having a condition or disease. Insome instances, exhibition of a positive immune response towards acondition or disease can be made from patient records, family history,and/or detecting an indication of a positive immune response. In someinstances multiple parties can be included in subject selection. Forexample, a first party can obtain a sample from a candidate subject anda second party can test the sample. In some instances, subjects can beselected and/or referred by a medical practitioner (e.g., a generalpractitioner). In some instances, subject selection can includeobtaining a sample from a selected subject and storing the sample and/orusing the in the methods disclosed herein. Samples can include, forexample, cells or populations of cells.

In some instances, treatments methods can include a singleadministration, multiple administrations, and repeating administrationas required for the prophylaxis or treatment of the disease or conditionfrom which the subject is suffering. In some instances treatment methodscan include assessing a level of disease in the subject prior totreatment, during treatment, and/or after treatment. In some instances,treatment can continue until a decrease in the level of disease in thesubject is detected.

The terms “administer,” “administering,” or “administration,” as usedherein refers to implanting, absorbing, ingesting, injecting, orinhaling, the inventive drug, regardless of form. In some instances, oneor more of the compounds disclosed herein can be administered to asubject topically (e.g., nasally) and/or orally. For example, themethods herein include administration of an effective amount of compoundor compound composition to achieve the desired or stated effect.Specific dosage and treatment regimens for any particular patient willdepend upon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Following administration, the subject can be evaluated to detect,assess, or determine their level of disease. In some instances,treatment can continue until a change (e.g., reduction) in the level ofdisease in the subject is detected.

Upon improvement of a patient's condition (e.g., a change (e.g.,decrease) in the level of disease in the subject), a maintenance dose ofa compound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained. Patients may,however, require intermittent treatment on a long-term basis upon anyrecurrence of disease symptoms.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Materials and Methods:

Compounds were tested in peroxide exposure neuronal cell model atCharles River Labs Discovery Research Services Finland as describedherein.

Cortical mixed cultures were prepared from E18 Wistar rat embryos(Laboratory Animal Center, Kuopio, Finland). The cortices were dissectedout and the tissue was cut to small pieces. The cells were separated by15-min incubation with DNase and papain. The cells were collected bycentrifugation (1500 rpm, 5 min). The tissue was triturated with apipette and the cells were plated (3×10⁵ cells/cm²) on poly-L-lysinecoated 48 wells in MEM supplemented with 2 g/l glucose, 2 mM glutamine,0.1 μg/ml gentamicin, and 10% heat-inactivated horse serum (HS-HI) andheat-activated bovine serum (FBS-HI). After three days in vitro, mediumcontaining MEM with supplements and 5% both sera was changed to thecells. On day 6 in vitro, the unwanted cell division was inhibited byadding cytosine arabinoside (10 μM final concentration) for 24 h. Thecultures were refed with MEM with supplements and 5% HS-HI beforeexperiments.

The wells in good shape were chosen for experiment on day 10 in vitro.Test compounds were diluted in MEM with supplements and 5% HS-HI. 24 hlater, 300 μM NMDA for 24 h was used as a control for total neuronaldeath, and H₂O₂ for 1 h was used to induce approximately 30-60% celldeath. DFX (100 μM) was used as a potential positive control forinhibition of H₂O₂-induced cell death. Wells treated with medium onlyserved as 0-control. Test compounds were pipetted to the cells 24 hbefore adding H₂O₂. After 60 min, the medium was removed and mediacontaining the compounds was pipetted to the wells for 24 h.

After 24 h the culture media of all wells was collected and possiblecell debris was removed by centrifugation (13 000 rpm, 3 min). A-100-μlaliquot was pipetted into a micro titer plate as duplicates, and equalamount of LDH reagent was pipetted to the wells. The absorbance at 340nm was measured immediately using a 3-min kinetic measurement protocolin Multiskan Ascent ELISA reader (Thermo, Finland). The change inabsorbance/min was determined, which is directly proportional to thereleased LDH (=cell death).

After the cell culture medium was collected for LDH measurement, 100 μlPrestoBlue solution (1:10 dilution of PrestoBlue reagent (Invitrogen,#A13261, lot 915815C) in culture medium) was added to the wells for 1 hincubation at +37° C. CO₂ incubator. Fluorescence at 560 nmexcitation/615 nm emission was measured using Victor fluororeader(PerkinElmer).

The number of wells per compound concentration used was 6 (n=6). One (1)concentration of 100 μM TUDCA and 1 mM 4-PBA separately and togetherwere studied. Statistical analysis were calculated on Microsoft excel.The values were analyzed two-tailed, equal variance t-tests comparingall conditions to all other conditions.

Example 1

The inventors tested TUDCA and 4-PBA in combination in a peroxideexposure neuronal cell model to explore what effects they may have inconcert. A peroxide exposure neuronal cell model was chosen for itscorrelation with oxidative stress and pathology in variousneurodegenerative diseases. In this study, the inventors evaluatedefficacy based on cell viability and cell death.

Use of TUDCA and 4-PBA, in combination, resulted in a 90.4% cellviability, a statistically significant improvement over control cellviability of 48.6% (p<0.003). (FIG. 1, Table 1) Individual treatmentswith TUDCA and 4-PBA resulted in cell viabilities of 59% and 72%respectively, not significantly different from control (p<0.414 andp<0.0.071 respectively). (FIG. 1, Table 1) The combination thereforecauses a 24% increase in efficacy over the sum efficacy of theindividual drug treatments. NMDA at 300 uM was used as a negativecontrol and resulted in about 0% cell viability. DFX provided theerratic result of about 11% less cell viability than the peroxideexposure condition. This result was thought to be due to DFX interferingwith the PrestoBlue measurement mode so this result was excluded.Efficacy is calculated as absolute difference in mean viability frommean viability of control.

The near return to control provided by the combination of molecules asseen in the PrestoBlue study is truly surprising and highlights thepotential for this combination as a therapy for diseases related toperoxide toxicity. The combination improved viability and decreased celldeath more than the sum of the benefits provided by each compound alone.

TABLE 1 Cell Viability Evaluated By PrestoBlue Following H₂O₂ ExposureAverage (%) Standard Error (%) No Treatment 100.00 2.21 H₂O₂ 40 uM 48.567.45 TUDCA 100 uM 58.59 9.10 4-PBA 1 mM 72.20 9.01 TUDCA + 4-PBA 100 um90.39 7.40 and 1 mM respectively DFX 100 uM 37.49 3.72 NMDA 300 uM 0.002.73

Use of TUDCA and 4-PBA, in combination, resulted in a 31.5% cell deathpercentage, a statistically significant improvement over control celldeath percentage of 62.5% (p<0.0.021). (FIG. 2) Individual treatmentswith TUDCA and 4-PBA resulted in cell death percentages of 60.4% and46.5% respectively, not significantly different from control (p<0.808and p<0.162 respectively). (FIG. 2) The combination therefore causes a71% increase in efficacy over the sum efficacy of the individual drugtreatments. DFX and NMDA were used as positive and negative controlsrespectively and resulted in 38.5% and 100% cell death respectively.Efficacy is calculated as absolute difference in mean viability frommean viability of control.

These surprising and synergistic results suggest the combination ofTUDCA and 4-PBA may be a novel, efficacious, and potent treatment toreactive oxygen metabolite-mediated oxidative damage, and therebyreducing neuronal oxidative stress and to treat at least one symptomassociated with, prevent the time of onset of, or slow the developmentof a disease associated with oxidative stress.

TABLE 2 Cell Death Evaluated By LDH Following H₂O₂ Exposure Average (%)standard error (%) No Treatment 0.00 1.25 H₂O₂ 40 uM 62.48 13.09 TUDCA100 uM 60.37 11.75 4-PBA 1 mM 46.47 11.05 TUDCA + 4-PBA 100 um 31.529.50 and 1 mM respectively DFX 100 uM 38.50 8.58 NMDA 300 uM 100.0016.90

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of treating a neurodegenerative disease,the method comprising administering to a subject: about 10 mg/kg toabout 50 mg/kg of body weight of a bile acid selected from the groupconsisting of tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid(UDCA), chenodeoxycholic acid, cholic acid, hyodeoxycholic acid,lithocholic acid, and glycoursodeoxycholic acid, or pharmaceuticallyacceptable salts thereof, and about 10 mg/kg to about 400 mg/kg of bodyweight of a phenylbutyric acid (PBA) selected from the group consistingof 4-phenylbutyric acid (4-PBA), glycerly(Tri-4-PBA),2-(4-methoxyphenoxy) acetic acid (2-POAA-OMe), 2-(4-nitrophenoxy) aceticacid (2-POAA-NO2), and 2-(2-naphathyloxy) acetic acid (2-NOAA), orpharmaceutically acceptable salts thereof, wherein the bile acid and thePBA are administered in an amount sufficient to treat theneurodegenerative disease.
 2. The method of claim 1, wherein the subjectis a mammal.
 3. The method of claim 1, wherein the subject is a human.4. The method of claim 1, wherein the neurodegenerative disease isselected from Multiple Sclerosis (MS), Alzheimer's Disease (AD),Huntington's disease (HD), Parkinson's disease (PD) Amyotrohic LateralSclerosis (ALS), Pick's Disease, Multi-Infarct Dementia,Creutzfeldt-Jakob's Disease, Dementia with Lewy Bodies (DLB), Mixeddementia, and Frontotemporal dementia.
 5. The method of claim 1, whereinthe neurodegenerative disease is caused by oxidative stress ormitochondrial dysfunction.
 6. The method of claim 1, wherein the bileacid and PBA are administered in an amount sufficient to reduce reactiveoxygen metabolite-mediated oxidative damage in a cell of the subject. 7.The method of claim 6, wherein the reactive oxygen metabolite-mediatedoxidative damage is hydrogen peroxide (H₂O₂) mediated damage.
 8. Themethod of claim 1, wherein the bile acid and PBA are administered in anamount sufficient to promote redox homeostasis in a cell of the subject.9. The method of claim 1, wherein the bile acid and PBA are administeredin an amount sufficient to reduce mitochondrial dysfunction in a cell ofthe subject.
 10. The method of claim 6, wherein the bile acid isadministered in an amount equivalent to about 10 mg/kg to about 30 mg/kgof body weight of TUDCA.
 11. The method of claim 6, wherein the PBA isadministered in an amount equivalent to about 10 mg/kg to about 100mg/kg of body weight of 4-PBA.
 12. The method of claim 6, wherein thePBA is administered in an amount equivalent to about 30 mg/kg to about100 mg/kg of body weight of 4-PBA.
 13. The method of claim 1, whereinthe bile acid and the PBA are administered separately.
 14. The method ofclaim 1, wherein the bile acid and the PBA are administeredconcurrently.
 15. The method of claim 1, wherein the bile acid and thePBA are administered once a day, twice a day, or three times a day.